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  • Q5® High-Fidelity DNA Polymerase


    Q5® High-Fidelity DNA Polymerase is a high-fidelity, thermostable DNA polymerase with 3´→ 5´ exonuclease activity, fused to a processivity-enhancing Sso7d domain to support robust DNA amplification. With an error rate > 100-fold lower than that of Taq DNA Polymerase and 12-fold lower than that of Pyrococcus furiosus (Pfu) DNA Polymerase, Q5 High-Fidelity DNA Polymerase is ideal for cloning and can be used for long or difficult amplicons. Q5 High-Fidelity DNA Polymerase is supplied with an optimized buffer system that allows robust amplification regardless of GC content. The 5X Q5 Reaction Buffer contains 2 mM MgCl2 at final (1X) reaction concentrations and is recommended for most routine applications. For GC-rich targets (≥ 65% GC), amplification can be improved by the addition of the 5X Q5 High GC Enhancer. Q5 High-Fidelity DNA Polymerase is unlike typical, lower fidelity PCR enzymes. To determine the optimal annealing temperatures for a given set of primers, use of the NEB Tm Calculator is highly recommended.

    Amplification of a variety of human genomic amplicons from low to high GC content using Q5 High-Fidelity DNA Polymerase. All reactions were conducted using 30 cycles of amplification and visualized by microfluidic LabChip® analysis.

    Product Source

    An E. coli strain that carries the Q5 High-Fidelity DNA Polymerase gene.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    Q5® High GC Enhancer5X
    Q5® Reaction Buffer-205X

    Advantages and Features


    • High-fidelity PCR
    • Cloning
    • Long or Difficult Amplification
    • High-throughput PCR

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 74°C.

    Storage Temperature


    Heat Inactivation


    Unit Assay Conditions

    25 mM TAPS-HCl (pH 9.3 @ 25°C), 50 mM KCl, 2 mM MgCl2, 1 mM β-mercaptoethanol, 200 μM dNTPs including [3H]-dTTP and 400 μg/ml activated Calf Thymus DNA.


    1. Anastassia Voronova Erin Coyne, Ashraf Al Madhoun, Joel V. Fair, Neven Bosiljcic, Catherine St-Louis, Grace Li, Sherry Thurig, Valerie A. Wallace, Nadine Wiper-Bergeron, and Ilona S. Skerjanc. (2013). Hedgehog Signaling Regulates MyoD Expression and Activity. J Biol Chem. 288(6), 4389–4404. PubMedID: PMC3567689
    2. Lieve Naesens, Luke Guddat, Dianne Keough, André B.P. van Kuilenburg, Judith Meijer, Johan Vande Voorde and Jan Balzarini (2013). ROLE OF HUMAN HYPOXANTHINE GUANINE 
      OF THE ANTIVIRAL AGENT T-705 (FAVIPIRAVIR). Molecular Pharmacology Fast Forward. 87247
    3. Hicham Bouabe (2013). A Protocol for Construction of Gene Targeting Vectors and Generation of Homologous Recombinant Embryonic Stem Cells. Methods in Molecular Biology . 1064, 337-354.


    1. What are the advantages to using Q5® High-Fidelity DNA Polymerase?
    2. What is the fidelity of Q5® High-Fidelity DNA Polymerase?
    3. How should I determine an appropriate annealing temperature for my reaction?
    4. What should my primer concentration be when using Q5® High-Fidelity DNA Polymerase products?
    5. How should I set up a PCR experiment using Q5® High-Fidelity DNA Polymerase?
    6. My template is GC rich or supercoiled. How can I optimize my product yield using Q5® High-Fidelity DNA Polymerase?
    7. Do I need to modify my annealing temperature when using the Q5® High GC Enhancer?
    8. When should I add the High GC Enhancer?
    9. Are the DNA fragments produced by Q5® High-Fidelity DNA Polymerase blunt-ended or do they have the single-base 3´ overhang that Taq DNA Polymerase yields?
    10. There is a precipitate in the bottom of the buffer tube. Is this normal?
    11. What length of product can be made by Q5® High-Fidelity DNA Polymerase?
    12. I am having trouble amplifying a template that is longer than 5kb. How can I optimize my product yield using Q5® High-Fidelity DNA Polymerase?
    13. Does Q5® High-Fidelity DNA Polymerase exhibit a strand displacement activity?
    14. Where can I find help troubleshooting my PCR?
    15. Will Q5® High-Fidelity DNA Polymerase incorporate dUTPs?
    16. I'd like to clone a fragment amplified with Q5® High-Fidelity DNA Polymerase. Do I have to blunt-end clone?
    17. Do other polymerases work in Q5® Reaction Buffer?
    18. I am competing in the iGEM competition. Do you have any products that I should consider purchasing from NEB?


    1. PCR Using Q5® High-Fidelity DNA Polymerase (M0491)


    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

    Selection Tools

    Feature Articles

    Usage Guidelines & Tips

    Troubleshooting Guides

    Interactive Tools

    Application Notes


    • Yonghe Zhang, Huiming Huang, Shanshan Xu, Bo Wang, Jianhua Ju, Huarong Tan, Wenli Li (2015). Activation and enhancement of Fredericamycin A production in deepsea-derived Streptomyces somaliensis SCSIO ZH66 by using ribosome engineering and response surface methodology. Microb Cell Fact. 14, 64. PubMedID: 25927229, DOI: 10.1186/s12934-015-0244-2
    • Binyamin D Berkovits, Christine Mayr (2015). Alternative 3' UTRs act as scaffolds to regulate membrane protein localization. Nature. , PubMedID: 25896326, DOI: 10.1038/nature14321
    • Jun Wu, Daiji Okamura, Mo Li, Keiichiro Suzuki, Chongyuan Luo, Li Ma, Yupeng He, Zhongwei Li, Chris Benner, Isao Tamura, Marie N Krause, Joseph R Nery, Tingting Du, Zhuzhu Zhang, Tomoaki Hishida, Yuta Takahashi, Emi Aizawa, Na Young Kim, Jeronimo Lajara, Pedro Guillen, Josep M Campistol, Concepcion Rodriguez Esteban, Pablo J Ross, Alan Saghatelian, Bing Ren, Joseph R Ecker, Juan Carlos Izpisua Belmonte (2015). An alternative pluripotent state confers interspecies chimaeric competency. Nature. , PubMedID: 25945737, DOI: 10.1038/nature14413
    • Longhai Dai, Can Liu, Yueming Zhu, Jiangsheng Zhang, Yan Men, Zeng Yan, Yuanxia Sun (2015). Functional Characterization of Cucurbitadienol Synthase and Triterpene Glycosyltransferase Involved in Biosynthesis of Mogrosides from Siraitia grosvenorii. Plant Cell Physiol. , PubMedID: 25759326, DOI: 10.1093/pcp/pcv043
    • Amin Zargar, David N Quan, Milad Emamian, Chen Yu Tsao, Hsuan-Chen Wu, Chelsea R Virgile, William E Bentley (2015). Rational design of 'controller cells' to manipulate protein and phenotype expression. Metab Eng. , PubMedID: 25908186, DOI: 10.1016/j.ymben.2015.04.001
    • Christine Henke, Pamela L Strissel, Maria-Theresa Schubert, Megan Mitchell, Claus C Stolt, Florian Faschingbauer, Matthias W Beckmann, Reiner Strick (2015). Selective expression of sense and antisense transcripts of the sushi-ichi-related retrotransposon - derived family during mouse placentogenesis. Retrovirology. 12, 9. PubMedID: 25888968, DOI: 10.1186/s12977-015-0138-8
    • Harish Nag Kankipati, Marta Rubio-Texeira, Dries Castermans, George Diallinas, Johan M Thevelein (2015). Sul1 and Sul2 Sulfate Transceptors Signal to Protein Kinase A upon Exit of Sulfur Starvation. J Biol Chem. 290, 10430-46. PubMedID: 25724649, DOI: 10.1074/jbc.M114.629022
    • Yuan Xue, Jossef Osborn, Anand Panchal, Jay L Mellies (2015). The RpoE Stress Response Pathway Mediates Reduction of the Virulence of Enteropathogenic Escherichia coli by Zinc. Appl Environ Microbiol. 81, 3766-74. PubMedID: 25819956, DOI: 10.1128/AEM.00507-15
    • Yafeng Li, Delu Song, Ying Song, Liangliang Zhao, Natalie Wolkow, John W Tobias, Wenchao Song, Joshua L Dunaief (2015). Iron-induced Local Complement Component 3 (C3) Up-regulation via Non-canonical Transforming Growth Factor (TGF)-β Signaling in the Retinal Pigment Epithelium. J Biol Chem. 290, 11918-34. PubMedID: 25802332, DOI: 10.1074/jbc.M115.645903
    • Bert De Rybel, Milad Adibi, Alice S. Breda, Jos R. Wendrich, Margot E. Smit, Ondej Novk, Nobutoshi Yamaguchi, Saiko Yoshida, Gert Van Isterdael, Joakim Palovaara, Bart Nijsse, Mark V. Boekschoten, Guido Hooiveld, Tom Beeckman, Doris Wagner, Karin Ljung, Christian Fleck, Dolf Weijers (2014). Integration of growth and patterning during vascular tissue formation in Arabidopsis Science. 345, 1255215. PubMedID: 25104393, DOI: 10.1126/science.1255215
    • Vidhyadhar Nandana, Sushant Singh, Abhay Narayan Singh, Vikash Kumar Dubey (2014). Procerain B, a cysteine protease from Calotropis procera, requires N-terminus pro-region for activity: cDNA cloning and expression with pro-sequence. Protein Expr Purif. 103C, 16-22. PubMedID: 25173974, DOI: 10.1016/j.pep.2014.08.003
    • Martin Kostovcik, Craig C Bateman, Miroslav Kolarik, Lukasz L Stelinski, Bjarte H Jordal, Jiri Hulcr (2014). The ambrosia symbiosis is specific in some species and promiscuous in others: evidence from community pyrosequencing. ISME J. , PubMedID: 25083930, DOI: 10.1038/ismej.2014.115
    • Xin Duan, Arjun Krishnaswamy, Irina De la Huerta, Joshua R Sanes (2014). Type II Cadherins Guide Assembly of a Direction-Selective Retinal Circuit. Cell. 158, 793-807. PubMedID: 25126785, DOI: 10.1016/j.cell.2014.06.047
    • Wang XJ, Zhang XJ, Hu W, Zhang TY, Wang SQ (2014). A simple and efficient strategy for the de novo construction of greater-than-genome-length hepatitis B virus replicons J Virol Methods. 207, 158-62. PubMedID: 25025817, DOI: 10.1016/j.jviromet.2014.07.009
    • Wilber Quispe-Tintaya, Ryan R White, Vasily N Popov, Jan Vijg, Alexander Y Maslov (2013). Fast mitochondrial DNA isolation from mammalian cells for next-generation sequencing Biotechniques. 55(3), 133-6. PubMedID: 24003945, DOI: 10.2144/000114077

    Quality Control

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Endonuclease Activity (Nicking):
      The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
    • PCR Amplification (Enhancer Dependent, GC-rich):

      The polymerase is tested in a polymerase chain reaction (PCR) using a GC-rich control template and specific primers, resulting in the enhancer-dependent production of the expected product.

    • PCR Amplification (Master Mix):
      The polymerase master mix is tested in a polymerase chain reaction (PCR) using a control template and specific primers, resulting in the expected product.
    • Protein Purity (SDS-PAGE):
      The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.

    Safety Data Sheet

    The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.


    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.